Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >350 million people and kills up t 1.2 million patients annually. Therapy primarily employs nucleos(t)ide analogs. These drugs profoundly suppress HBV but they rarely cure patients due to incomplete inhibition of viral replication. Furthermore, control of HBV often fails due to evolution of drug resistance mutations. Pharmacologically curing HBV in more patients should be possible by suppressing HBV further with new drugs to be used in combination with the nucleos(t)ide analogs. HBV reverse transcription requires 2 viral enzymatic activities that are both located on the viral reverse transcriptase protein: the DNA polymerase that is targeted by the nucleos(t)ide analogs and the ribonuclease H (RNAseH) that destroys the viral RNA after it has been copied into DNA. Anti-HBV RNAseH drugs have not been developed because enzyme suitable for drug screening could not be made. We recently made active HBV RNAseH and identified 13 inhibitors of the RNAseH. HBV has 8 genotypes (A-H), and anti-RNAseH drugs must inhibit a wide range of HBV strains to be clinically effective. We found that RNAseH inhibitors identified against genotype D and H isolates can block replication of a genotype A isolate, so cross-genotypic inhibition is possible. However, the genotype D and H RNAseH isolates we tested are differentially sensitive to RNAseH inhibitors. We do not know whether these dissimilarities are due to genotype-specific or isolate-specific differences because have examined only 1 isolate for each genotype. Furthermore, we have not characterized recombinant RNAseH from genotypes B and C, which are the most medically relevant genotypes. In this Small Research Grant (R03) project, we will evaluate the degree to which HBV's high genetic variation affects its sensitivity to RNAseH inhibitors. Aim 1: Evaluate how differences between and within HBV's genotypes affect sensitivity to RNAseH inhibitors. We will test sensitivity of variant HBV RNAseH sequences from genotypes B, C, and D to a set of RNAseH antagonists in biochemical and viral replication assays. Aim 2: Determine how nucleoside analog resistance mutations affect sensitivity to RNAseH inhibition. We will introduce common nulceos(t)ide analog resistance mutations into HBV genotype B, C, and D isolates and evaluate how they affect sensitivity of viral replication to an RNAseH inhibitor. This project will: 1) Reveal whether HBV genotypic differences will complicate anti-RNAseH drug development; 2) Help determine whether communication between the 2 active sites on the reverse transcriptase may complicate combination therapy with nucleos(t)ide analogs and RNAseH inhibitors; and 3) Generate a set of variant HBV RNAseHs for screening drug candidates for cross-genotypic efficacy.